The dissociation of gaseous polypeptide ions, in a tandem mass spectrometry experiment, plays a role in several commonly used approaches for the identification of proteins. The most commonly used approach for activating polypeptide ions has involved energetic collisions with neutral target gases, and is referred to as collisional activation.
A range of collisional activation conditions have been utilized that include collision energies ranging from a few electron volts to as high as several kilo-electron volts; numbers of collisions ranging from a single collision to many hundreds of collisions; and, time-scales ranging from the time for a single collision to hundreds of milliseconds. In general, collisional activation methods have been useful in deriving primary structure information from peptide and protein ions. However, no single dissociation method has able to provide all structural information of interest. For example, collisional activation often fails to provide complete primary structure information, and often fails to provide information regarding the positions of post-translational modifications.
Approaches other than energetic collisions with gaseous targets have been also examined for dissociating polypeptide ions. These include, for example, collisions with surfaces, referred to as surface-induced dissociation, and a range of photo-dissociation techniques, such as infra-red multi-photon dissociation (IRMPD), black-body infra-red dissociation (BIRD), and single-photon UV-photo-dissociation at one of several wavelengths. In the case of multiply-protonated polypeptides, ion-electron and ion-ion reactions have been used. Electron capture by, or electron transfer to, a multiply-protonated peptide gives rise to fragmentation that is often highly complementary to that resulting from collisional activation. The former is referred to as electron capture dissociation (ECD) and the latter is referred to as electron transfer dissociation (ETD). Both ECD and ETD have proven to be of particular utility for the characterization of post-translationally modified peptide and protein cations.
In the case of an ETD experiment, products from an ion/ion reaction can be allocated into one of three principal categories. These are proton transfer, a competing ion/ion reaction that generally does not lead to fragmentation; electron transfer followed directly by dissociation (i.e., the ETD process); and, electron transfer without subsequent dissociation of the polypeptide product. The partitioning between these three reaction categories is, at least to some extent, particular to each species of reactant ions. For example, the competition between proton transfer and electron transfer is known to depend strongly upon the identity of the reagent anion. The size and charge state of the peptide ion may play a significant role in determining the extent to which ETD occurs relative to electron transfer without dissociation, and the temperature of the bath gas in the electrodynamic ion trap used as a reaction vessel can affect the relative contributions of the total ETD, the relative contributions of individual reaction categories that contribute to ETD, and the extent of electron transfer without dissociation. Doubly-protonated peptides of the size often observed from tryptic digests, for example, usually show significantly less ETD than the triply charged versions of the same peptide.
From an analysis perspective, it may desirable to minimize the competitive proton transfer channel and to maximize ETD relative to electron transfer without dissociation. The selection of the reagent anion may be important in this regard. One technique that may maximize the extent of ETD for species that undergo electron transfer, but do not dissociate, is to subsequently activate these electron transfer products. That is, subsequent activation of the electron transfer (ET) “survivors” can improve the net conversion of precursor ions to structurally informative product ions.
In this regard, it may desirable to maximize the dissociation of the survivors while minimizing dissociation of proton transfer products. The latter species generally give rise to b-and y-type ions that could complicate spectral interpretation and may compromise the quality of data-base matching algorithms that assume the formation of only the c-and z-type ions generally associated with ETD. The use of elevated bath gas temperature is one technique of altering the extent of ETD. However, this approach can affect both the reactant ions and the product ions and, therefore, may not be exclusively an activation method for survivor ions.
The use of elevated bath gas temperatures, for example, has not been shown to consistently provide improved ETD yields relative to the use of room temperature bath gas.